Figure 3.

LAT follows the Rab6/Syntaxin-16–dependent canonical retrograde transport from endosomes to the TGN. (A–F) Jurkat cells expressing both GalT-GFP-SNAP and HA-LAT were incubated at 4°C with anti-HA Ab, washed, and incubated at 4°C with (+BG) or without (−BG) BG-PEG9-NHS. After washing, cells were incubated at 37°C for 4 h (A and B) or activated on slides for 30 min with Raji cells (C–F) left unpulsed (−SEE) or pulsed with SEE (+SEE). Immunolabelings were performed using anti–mouse Ig (Alexa Fluor 568) to label the anti-HA Ab and anti-GFP to label the GalT-GFP-SNAP. Colocalization of anti-HA and GalT-GFP-SNAP is shown. Quantifications show Mander’s colocalization coefficient (B, D, and F). (B) Quantification of the colocalization after 4 h of antibody uptake. (D) Quantification compare colocalization in cells stimulated for 30 min with unpulsed or SEE-pulsed Raji B cells. (F) Quantification of the colocalization in Jurkat T cells, expressing a control (ShC), Rab6-specific (Sh6), or Syntaxin-16–specific shRNA (Sh5), activated with SEE-pulsed Raji B cells. Images show the maximum intensity from z-projections of three to five z-stacks covering the Golgi apparatus. Insets show the Golgi compartment. The profile plots of RGB images from ImageJ are shown. Means, n = 3 (A and B), 2 (C and D), and 2 (E and F) independent experiments for each condition. Bars, 5 µm. ****, P < 0.0001. (B) Student’s t test. (D and F) One-way ANOVA.